Obesity is associated with development of metabolic syndrome, non-insulin dependent diabetes mellitus (NIDDM), and cardiovascular diseases such as hypertension. Adipocytes play a central role in the physiological consequences of the energy imbalance inherent to obesity. Formation of adipocytes depends on the peroxisome proliferator-activated receptor gamma (PPAR&#61543;), a protein that functions as the [unreadable]master switch[unreadable] in regulating the production of other proteins needed for lipid and carbohydrate metabolism in adipocytes. PPAR&#61543;is also the cellular target of a commonly prescribed class of anti-diabetic drugs, the thiazolidinediones (TZDs). These drugs alter PPAR&#61543;activity and PPAR&#61543;protein levels, an indication that understanding the link between PPAR&#61543;activity and PPAR&#61543;protein stability may offer new insights into how obesity contributes to NIDDM. PPAR&#61543;stability in adipocytes is regulated by enzymes of the ubiquitin proteasome pathway, a highly selective signaling pathway that targets proteins to the proteasome by tagging the protein with multiple ubiquitin polypeptides. Our preliminary studies also show that the region of PPAR&#61543;responsible for binding TZDs contains a signal for binding to ubiquitin and that a functioning ubiquitin system is necessary for TZDdependent regulation of PPAR&#61543;activity in adipocytes. Using siRNA-based screening, we identified an ubiquitin ligase, Siah2 that regulates PPAR&#61543;protein levels in adipocytes. Siah2 expression is increased during adipocyte formation and is abundant in adipose tissue. Moreover, Siah2 is required for insulin-stimulated glucose uptake in 3T3-L1 adipocytes, potentially linking modulation of PPAR&#61543;protein stability by the ubiquitin system to regulation of insulin sensitivity. We hypothesize that Siah2-dependent regulation of PPAR&#61543;protein levels is a determinant of PPAR&#61543;activity and insulin signaling in adipocytes. Our goal is to provide mechanistic insight into the role of Siah2 in controlling the relationship between the ubiquitin proteasome pathway, PPAR&#61543;activity, and insulin sensitivity in adipocytes. In specific aim 1, we will use 3T3-L1 adipocytes to determine if Siah2 regulates the ubiquitin proteasome-dependent degradation of PPAR&#61543;and TZDdependent activation of PPAR&#61543;via direct interaction with PPAR&#61543;. In specific aim 2, we will use 3T3-L1 adipocytes and murine models to examine the role of Siah2 in adipogenesis and insulin signaling in adipocytes. Specific aim 3 will use a murine model of obesity to focus on the effect of diet-induced obesity and insulin resistance on Siah2 expression and ubiquitin proteasome activity in adipose tissue. The proposed study will provide new insight into how PPAR&#61543;activity is regulated in adipocytes. Our long-term goal is to define the mechanisms underlying the role of PPAR&#61543;in glucose and lipid metabolism and to identify potential novel therapeutic targets in the treatment of obesity and type 2 diabetes through studies of ubiquitin proteasome regulation of PPAR&#61543;activity and expression in adipocytes.